Deciphering the antimicrobial, antibiofilm and membrane stabilizing synergism of Mikania scandens (L.) Willd. leaves and stems substantiation through in vitro and in silico studies

Highlights · Multidrug-resistant bacterial infections as well as associated diseases are one of the ever-growing concerns of the modern world.· Mikania scandens like traditional natural affluence of such diseases can be the best alternative.· Leaves of Mikania scandens corroborated its ethnomedicinal infectious utility against a panel of microbes along in inflammation.· Notably, this same parts comprised exorbitance activity against carbapenem resistant Pseudomonas aeruginosa as evident to MIC, MBC and biofilm inhibition.· This antibacterial and antiinflammatory resilience was further affirmed using computational investigations.


Introduction
Irresponsible and uncontrolled use of antibiotics resurge the evolution of multi drug resistant (MDR) pathogens which imparts a serious havoc for human races across the globe through hatching plaguing like exigency that culminate the morbidity and mortality rates indicatively [1]. That genesis of MDR meanwhile prospected in Staphylococcus aureus as methicillin resistant S. aureus (MRSA) and pan-resistant Pseudomonas aeruginosa strain emerged major apprehension for its infectivity [2]. Traditional antibiotics' mode of action against these pathogens were inhibition of planktonic bacterial cell growth instead of focusing on the naturally occurring mode of bacterial growth, known as biofilm, which formulated an exopolymeric physical layer on surroundings which resists the pathogens from its unfavorable environment and contributes to recurrent infections likely periodontosis, cystic fibrosis, chronic prostatitis, endocardititis and antibiotic resistance [3]. Over 60% of microbial illnesses may be induced by biofilms, which also account for two-thirds of all bacterial infections in humans [4]. Therefore, inflammatory mediated pathogenic infections is one of the major factor for tumor progression which subsequently aftermath to the apparition of cancer such pathogens likely Helicobacter pylori for gastric cancer, Chlamydia psittaci for ocular lymphomas and Salmonella typhi for hepatobiliary carcinoma [5,6]. On account of Center for Disease Control and Prevention (CDC), around 20000 fatalities worldwide were attributable to pathogenic resistance among the 2 million reported case [7]. By 2050, it is predicted that there would be 10 million more fatalities globally due to antimicrobial resistance, with a treatment price tag of almost $100 trillion [8]. Pharmacological target for the tackling this pathogenic infections, biofilm inhibitory antibiofilm medications should be the subject of intensive study [9]. Various green nonlethal methods for controlling biofilms have been developed recently, but none have yet demonstrated efficacy due to the fact that the new antibiofilm medications' mode of action is substantially less vulnerable to the development of resistance [10]. The exploration of medicinal plants utilizing cutting-edge technology is currently being reevaluated as safe conventional treatments for pathogenic infections and as a practical method for identifying novel antimicrobial bioactive molecules to address this pervasive public health issues [11]. Researchers are seeking for natural compounds that have antibacterial action and work in synergy with currently available antimicrobial therapies since microbes that are resistant to conventional treatments are becoming more prevalent. As a result, herbal remedies are emerging as a promising alternative to conventional infection treatment.
Mikania scandens (L.) (MS) is a twining plant that can be found growing as a weed throughout the country and locally known as "Jarmany lota" in Bangladesh [12]. Intersecting analysis of various literature unveiled that leaf of this plant traditionally used in several part of the globe as remedial of wounds, bruise, itching and headache [13][14][15][16]. This pain modulation affluence therefore scientifically verified [17] along disclosing antioxidant [18], anti-inflammatory [19], antidiabetic [20], thrombolytic [21] and neuropharmacological [22] potency of MS. Mikanolide, a sesquiterpene lactone metabolite, has been isolated from this MS plant so far [23].
In this study, the information gleaned herein is used as baseline to define the MS plants' leaves and stems antimicrobial and antbiofilm activities as well as their synergism with regard to anti-inflammatory potentialities using a range of in vitro and in silico screening methodologies.

Collection of plant materials
Fresh mature plant part stems and leaves of Mikania scandens (L.) were collected from the Chittagong University campus. Thereafter, this plant was authenticated by one of the countries prominent taxonomist Prof. Dr. Shaikh Bokhtear Uddin, Department of Botany at the University of Chittagong. Subsequently, following standard herbarium techniques one of the voucher specimen had been deposited in the Herbarium of Chittagong University (HCU) for future reference. The samples (leaves, stems) were cut into small pieces (1-2cm), washed thoroughly using distilled water, air-dried at room temperature, and pulverized to fine powder mechanically. During this precautions taken to avoid admixture of each of the plant parts.

Preparation of extracts
The fine powdered of each of the plant parts (leaves and stems) samples were sequentially extracted with polar and non-polar solvents based on solvents polarity index (Petroleum ether, Carbon tetrachloride, Chloroform, Ethyl acetate, and Ethanol). Whereat 200g powder was amalgamated with 500 ml solvents for extraction in a dark vessel and kept it there for 5 days with manual application of gentle shaking. Then, the extracts were filtered using Whitman filter paper and concentrated at low temperatures (40-50 • C) by a rotary vacuum evaporator. Thus, the extract obtained was termed crude extract. The crude extract of each sample was weighed and calculated the yield percentage of each sample as follows: Here, W 1 = Net weight of powder in grams after extraction. And W 2 = Total weight of wood powder in grams taken for extraction.

Test organisms
All of the microorganisms used in the tests were taken from culture collection stocks maintained by the Molecular Biology Lab of Department of Microbiology at the University of Chittagong

Fungal strains
The fungus Candida albicans ATCC10231, Aspergillus niger ATCC16404, Aspergillus fumigatus and Penicillum sp. were culled for antifungal properties evaluation of MS crude extracts of several solvents.

Determination of antimicrobial activity
The antibacterial and antifungal activities of the plant extracts were done by disc diffusion method and poisoned food technique respectively [24,25]. Wherein, Nutrient agar (NA) was used to culture the test bacteria; Muller-Hinton Agar (MHA) medium was used for disc diffusion method and Potato Dextrose Agar (PDA) medium was used for the culture of fungi with the exception of Candida albicans, whose nutritional needs and plate development are very similar to those of bacteria. A 4 mm size Whatman filter paper disc composed 10 µl of each plant extract integrated on the LB media plates for bacteria with having 1.6 × 10 7 CFU/ml organismal strain and PDA plates with 1.7 × 10 8 CFU/ml fungal strain. Thereby, those bacterial and fungal plates were incubated at 37 • C and room temperature for 24h following respectively after 4h fridge storage at 4 • C for dissemination of extracts.

Phytochemical analysis of mostly active extracts
The extracts were tested for significant phytochemicals according to recognized procedures. Several chemical assays utilizing accepted techniques were used to screen the aforementioned medicinal plant extracts for diverse phytochemical components [26,27].

Determination of MIC and MBC
A two fold macro-broth dilution method in accordance with Clinical and Laboratorial Standards Institute was employed to ascertain the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) of the ethyl acetate extracts of MSL and MSS [28]. Each tube was inoculated with 1 ml of diluted bacterial (1.6 × 10 7 CFU/ml) and fungal (1.7 × 10 8 CFU/ml) suspension, each of which included 1 ml of LB (for bacteria) and Sabouraud (for fungi) broth, as well as a variety of test materials with concentrations ranging from 500 to 2500 µg/ml. Following that, both tubes had a 24h incubation period-for bacteria, at 37 • C, and for fungus, at room temperature.
A suspension aliquot of 0.5 ml was placed into pre-sterilized petri dishes with MHA and PDA medium in order to explore the potential MBC, and MFC of antimicrobial agent in broth culture tubes where the microbial growth was not visible. After that, the plates were incubated for 24h at 37 • C for bacteria and room temperature for fungal inoculum in order to observe the microbial growth. MBC and MFC were defined as the maximum dilution at which at least 99% of bacteria were inhibited. Testing was undertaken in triplicate for each extract concentrations.

Time kill bacterial susceptibility to MSL and MSS extracts
Time-kill bacterial sensitivity to extracts was examined, according to Ajiboye et al (2016) description [29]. In Luria Bertani (LB) medium, bacterial cells were cultured during the night. Afterward, the cells were collected by centrifugation and resuspended in 50 mL of new LB media to produce an OD600 = 0.1 in a 250 mL conical flask. After that, the cells were grown aerobically at 37 • C in a shaker incubator. 15ml of culture were divided among 3 conical flasks at mid-log phase (OD600 = 0.5). After that, an antibiotic (ciprofloxacin) was added as a positive control, 0.5% dimethyl sulfoxide was added as a negative control, and crude extract of the tested organisms at MBC concentration. After that, the mixes were incubated for 3 hours at 37 • C.
Every 20 minutes for the first three hours of incubation, the incubation mixture's absorbance was measured at 600 nm. Samples of the control cultures and the extract-treated cultures were taken for colony formation at intervals of 0, 20, 40, 60, 80, 100, 120, 140, 160, and 180 min, respectively, and centrifuged to collect the cells as a pellet. After being thoroughly cleaned, the cells were diluted with 0.9% NaCl, combined with molten soft LB-agar at 42 • C, and then put onto agar plates with solid LB-agar. The plates were then housed in an incubator at 37 • C. Colonies were counted 24 hours later.

Anti-biofilm activity test
The crystal violet staining technique, as significantly modified by Christensen et al. (1985), was used to assess the impact of test extracts on biofilm development [30]. Bacterial cultures were performed using LB broth medium. Each test tube received 0.5ml of plant extract (0.5× MIC, 1×MIC, and 2×MIC). The negative control was considered to be equal amounts of water. Each test tube received a final volume of 1ml after 0.5ml of bacterial suspension (from a 24-hour culture) was added. As an extra nutrition, sterile LB was also supplied to each tube. Following that, the test tubes were kept at 37 • C for three days to facilitate the cell adherence.
The test tubes were cleaned five times with sterile distilled water after incubation to get rid of any bacterial cells that were loosely adhered. Following drying, the tubes were stained with 1% crystal violet. The tubes were rinsed to remove extra stain after 15 minutes. The test tube was once again destaining with 2 ml of 90% ethanol. At 530 nm, the destained solution's absorbance was quantified. Percentage of biofilm inhibition was measured using the following equation:

Membrane stabilization test
The techniques of hemolysis caused by a hypotonic solution were used to scrutinize the membrane stability of extracts in vitro antiinflammatory effectiveness using the human red blood cell (HRBC) membrane stabilization procedure of Vuna & Botting (1995) [31].
Six individuals provided their fresh whole blood, which was then combined with an equivalent amount of sterilized Alsever solution and centrifuged at 3000 rpm for 10 minutes. The packed cells were then washed three times with isosaline before being made into a 10% v/v suspension using isosaline. Hypotonic solution (5 ml) and erythrocyte suspension (0. 5 ml) were combined. Next, the extractives (25-200 µg/ ml) and a conventional medication (Diclofenac sodium) were applied to certain samples and not to others. Extracts and drugs were not introduced to the control group. Following a 10-minute incubation period, the mixture was centrifuged at 3000 rpm for 20 minutes to determine the amount of hemoglobin. After the supernatant was decanted, the hemoglobin content was calculated at 560 nm. %ofhemolysis = Theopticaldensityoftestsamples Theopticaldensityofthecontrol × 100

Acquisition of compounds and theirs pharmacokinetic properties
From M. scandens leaves, the literature review compiled 23 (Table 8) previously discovered active components [12]. With the aid of PubChem (https://pubchem.ncbi.nlm.nih.gov/) and SWISSADME (http://www. swissadme.ch/), the information about the gathered compounds and their drug similarity characteristics were retrieved and computed. SWISSADME filtered the drug-likeness distinctive features of the active components from M. scandens leaves using compound canonical SMILES from the PubChem database based on Lipinski's rule of five [32].

Ligands and targets modulation for molecular docking
Potent ligands discovered through drug likeness criteria, and the reference drug ciprofloxacin and sertaconazoles were all downloaded in .sdf format from the PubChem chemical library. Additionally, the Lig-Prep tools prepared the compounds for molecular docking investigations that embedded to Schrodinger's suite-Maestro v12.5 using the procedures we previously outlined [33]. The crystal structures of each target protein were gathered from the RCSB Protein Data Bank in order to optimize and minimize the each structures using another integrated tools protein preparation wizard. With OPL3S force field, all charges and bond orders were assigned, heavy atoms were allocated hydrogen, selenomethionine was substituted out for methionine, water was removed [34].

Standard precision molecular docking by Glide
To create receptor grids and carry out flexible molecular docking tests, Glide, an add-on for Schrodinger Suite-Maestro version 12.5, was utilized [35]. The force field of OPLS3 was used to generate a grid for each protein using the default settings of van der Waals scaling factor 1.00 and charge cut-off value 0.25. A cubic box of 14 Å × 14 Å × 14 Å size was generated over the active site (co-crystallize ligand site) pockets of each protein. The best scoring pose and docking value about each ligand were individually recorded throughout the docking assays, whom were completed using Glide's Standard Precision (SP) scoring system.

Yielding percentage of MSL and MSS extractives
The yielding percentage of extractives by different solvents Petroleum ether, carbon tetrachloride, chloroform, ethyl acetate and ethanol were 1.7%, 4%, 2.4%, 14.5% and 23.3% respectively from MSL and 0.9%, 5%, 1.65%, 8.9% and 27.5% respectively from MSS. That clearly indicated that the quantity of polar molecules was far higher than that of non-polar elements.

Qualitative antimicrobial activity test of MSL and MSS extractives
Variable non-polar (petroleum ether, carbon tetrachloride, chloroform) and polar (ethyl acetate and ethanol) solvent extracts from Mikania scandens stems (MSS) and leaves (MSL) had been exerted to ordain the antimicrobial efficiency against eleven human pathogenic organisms and five carbapenem-resistant isolates from burn wound infections in disk diffusion assay. The most practical approach for determining whether microbes are susceptible or resistant to different antimicrobial agents is the disc-diffusion method (DDM); nevertheless, the accuracy of this method depends on the upkeep of standard operating protocols [36]. Whereas, by the use of MSL extracts against bacterial pathogen, non-polar solvents showed an inhibitory zone of 9 to 23 mm that was somewhat larger than the polar extracts' 8 to 22 mm zone. On them, the petroleum ether and ethyl acetate extracts were the foremost one toward K. pneumoniae and P. aeruginosa which explicitly parallelism to standards ciprofloxacin effects. This ethyl acetate extracts supremacy therefore observed in aerial part extract of this same plants constructing the highest 9.37 mm inhibitory zone which articulately preceded by present studies [37]. Likewise, this research emphasized the furthest zone of inhibition that over some plant extracts displayed [38][39][40]. In the case of fungi, Ethyl acetate extracts from polar solvents conferred a much larger 24 ± 1.6 mm inhibitory zone against fungus Candida albicans when compared to non-polar carbon tetrachlorides 18 ± 1.25 mm zone. That furthest zone diameter whither pointedly over than sertaconazoles 22 ± 1.7 mm zone in diameter (Table 1). Contrariwise, Sh. dysnteriae resistant to all the extracts of MEL. Other hand, MSS non-polar extracts exhibited lower sensitivity against five bacterial (out of 10) and one fungal pathogens with subordinate zone of inhibition exception to E. coli's 20 ± 1.3 mm zone by petroleum ether. Conversely polar ethyl extracts of stem consistent to its leaf Note: 4mm in diameter paper disc soaked with different extracts; here, bold indicated highest zone of inhibition and (-) means no inhibition. *Fungal pathogen.    1) and MSS (Fig. 2) further evaluated for antimicrobial susceptibility at two different concentrations of 500 µg/disc and 1000 µg/disc. MSL at both concentrations (at 1000 µg/disc= 20 -35 mm and at 500 µg/disc= 12 -23 mm inhibitory zone) showed dose dependent antimicrobial activity for all organisms tested here including carbapenenm resistant microorganisms except E. coli at 500 µg/disc concentration. This extracts notable affluence potency against S. aureus meanwhile preserved in here with 20 mm inhibition zone at 1000 µg/disc in both types of strains. Whereas, comparatively less effectiveness of ethyl acetate MSS than its MSL, correlated the precedent findings containing an array of 7 -15 mm inhibition zone at the greatest concentration along resistance to E. coli and V. cholera. This contextual fact therewithal seen against carbapenem resistant microbes with portraying almost entire panel of microbial resistance. Table 4 displays the percentage of the fungal radial mycelial growth that was inhibited. At the concentration of 50 and 100 µg/ml, the ethyl acetate MSL extract may completely inhibit the Penecillium sp., A. niger that was cabbalistically outdo the standard sertaconozols inhibition. Conversely, A. fumigatus exhibited less inhibition than other species. This new threshold for maximal inhibitory doses of 100 µg/ml surpasses the previous studies on the antifungal properties of plant extracts [46][47][48].   [37]. Two of current plants generic lineages M. laevigata and M. glomerata's further substantiated its antibacterial action whereat non-polar solvent hexane extract defined bacterial growth inhibition and death at MIC of 12.5 to 100 mg/ml and MBC of 25 to 400 mg/ml concentrations respectively as indicative of lower polarity bioactive substances which is opposed to current findings [50]. This positive reactiveness of this extracts might be attributed through ATP hydrolysis, coagulation of cytoplasm, altering proton motive force, inhibiting DNA gyrase, protein synthesis, and cell wall permeability [51] and the difference in the MIC and MBC owing to variation in phytoconstituents nature [52]. A number of plant derived alkaloids and phenolic substances comprehensively picturesque theirs antimicrobial robustness restraining the fasten efflux of possible drug candidates that raising the permeability of bacterial cell wall to reach the intrabacterial cytoplasm so that refraining of the ribosomal protein synthesis takes place [53]. Another report also hypothesized the involvement of polyphenols (phenolics, tannins, flavonoids) in the cell membrane and cell wall through forming hydrogen bond within hydroxyl group and enzyme penetrating into cell and doing coagulation of cellular contents [54]. Notwithstanding that bacterial structural alterations of efflux and influx system for resistance documented in S. aureus, P. aeruginosa and A. baumannii [55,56]. Importantly, this same ethyl acetate MSL extracts disclosed almost considerable eptitude against both gram positive and negative bacteria opposed to conventional plant extracts resistance towards gram-negative bacteria due to hydrophilic cover over peptidoglycan which accountable for easy accessible of plant extractives in gram positive bacteria [57]. Note: (+) indicates the presence of growth and (-) means the absence of growth and ND means not detected up to 2500 µg/ml in MIC and MBC was not found up to 5000 µg/ml. Note: (+) indicates the presence of growth and (-) means the absence of growth and ND means not detected up to 2500 µg/ml in MIC and MBC was not found up to 5000 µg/ml.
Conjointly, resistance to carbapenem workable by mediating β-lactamase and hydrolytic enzymes which each molecules capable of hydrolyzing 10 3 β-lactam ring per second (drug substance/s) resultant to 100 million molecules elimination in each second with 10 5 enzymes [58,59]. On those resistant pathogens, ethyl acetate MSL extracts showed dominant sensitivity effect of bacteriostatic and bactericidal with two gram-negative (P. aeruginosa and A. baumannii) carbapenem resistant medical isolates composing 2000 -5000 µg/ml concentrations of MIC and MBC along MBC/MIC ratio between 2 -2.5 clarify theirs bactericidal notion of the extracts (Table 5). Whither, MSS posed only bacteriostatic potential against A. baumannii at 2000 µg/ml and persist resistance to other bacterial strains at both MIC and MBC levels. That forcible feasibility against gram-negative bacteria meantime concurrent to antecedent findings. This homologous dominancy also found in one of its generic predecessor M. micrantha leaves whereat deoxymikanolide isolated from that plant part exhibited the lowest MIC and MBC value of 62.5 and 125 mg/L respectively against Xanthomonas campestris pv. vesicatoria and Xanthomonas campestris pv. citri [60]. Presumably, inhibition of enzymatic modulation and degradation of drug molecules, and probable inhibitors of major facilitator superfamily of efflux pump (EP) tangibly RND (resistance-nodulation division) superfamily of gram-negative bacteria were the functional mode for this MSL extract as per literature reported about carbapenem resistance mechanistic target attribution [61,62]. From Table 7, it was found that the minimum concentrations needed for the fungicidal activity of MSL ethyl acetate extract were 100 µg/ml against Penicillium spp. and 200µg/ml against A. niger and A. fumigatus. However, the MIC values were 50 µg/ml for Penicillium spp. and 100 µg/ ml for A. niger and A. fumigatus. Theirs, MFC/MIC proportion was also within 2 as indicative of strong microbiocidal activities. Moreover, Siddiqui et al. (2013) found that the ethyl acetate extract of MSL revealed effective antifungal activity against F. oxysporum, P. graminicola and R. solani with MIC values of 250-500 µg/ml on phytopathogenic fungi [12].

Time-kill bacterial susceptibility with MSL and MSS extractives
One gram negative and gram positive bacteria, namely E. coli and B. cereus, respectively, were chosen for the time kill sensitivity assay on the basis of ≤4 MBC/MIC ratio with the most efficient ethyl acetate MSL extracts.
When compared to bacteria that were just exposed to DMSO, the absorbance was lowered when E. coli and B. cereus were treated with an ethyl acetate extract of MSL at bacterial MBC concentration. Similar changes in these bacterial cells' absorbance were brought about by the reference antibiotic Ciprofloxacin. A bacterial cell can be killed, as this study's results shown, with the passage of time. Thus according to Fig. 3A, ethyl acetate MSL extract reduced bacterial cell absorbance at 0.30 almost equivalent to reference drugs absorbance rate and nearly killed E. coli in the period of 160 to 180 minutes. It was discovered in Fig. 3B that extracts could not completely kill bacterial cells at the same rate as antibiotics alone to kill gram-positive B. cereus in 180 minutes. The cause of their presence of a thick peptidoglycan coating may take a long time to eradicate. This noteworthy antibacterial capacity was investigated against gram-negative bacteria that were atypical for plant extractives to have microbiocidal affluence, this large response was indeed compatible with quantitative antimicrobial screenings of ongoing investigations. In a study by Ajiboye et al. using a similar timekill susceptibility experiment aligned with our findings, the researchers noted that the extract lowered absorbance relative to bacterial cells treated using simply DMSO [63]. Euclea crispa ethyl acetate leaf extracts elucidated complete inhibition of cells in 120 min against gram negative K. pneumoniae at 2×MIC concentrations [64]. Note: (+) indicates the presence of growth and (-) means the absence of growth.
Here bold indicates the lowest MIC and MFC value.

Antibiofilm properties of MSL and MSS extractives
Without the evaluation of antibiofilm potentiality, only MIC determination was not noteworthy because of biofilms microorganism extracellular polysaccharides matrix resulting into microbe strong attachment to biotic/abiotic surfaces which trigger the efflux pumps to expel the drugs or lowering the drugs penetration into the cell [65,66]. Consequently make those drugs molecules resistant to those bacterial strain communities and can spread this resistance genetic material among communities other members. As per past record of E. coli, K. pneumoniae, P. aeruginosa, and S. aureus microbes ability to form biofilm [67], we had been utilized these carbapenem resistant pathogens isolated from burn wound infections to divulge the antibiofilm activity of our studies prominent ethyl acetate MSL extracts. These extract at the concentration of half of MIC inhibited a little biofilm formation but in MIC concentration it inhibited 50% biofilm formation occurred by E. coli. and inhibited less than 50% for other pathogens (Fig. 4). However, it prevented about 80% of biofilm formation in gram-negative P. aeruginosa and E. coli at 2×MIC concentration which in line with this extracts earlier findings about robust efficiency against those gram staining bacteria, even though it was around 60% in S. aureus and K. pneumoniae. As comparison to MIC concentrations of MSL, isolated asimicin molecules from Annona senegalensis seeds visualized almost comparable biofilm eradication of 36.5 and 43.2% with S. aureus and E. coli [68]. Contrariwise, methanolic Carum copticum extract demonstrated 25 -60% biofilm inhibition with gram negative (E. coli, K. pneumoniae, P. aeruginosa and A. baumannii) and 20 -30% inhibition in gram-positive bacteria (S. aureus and B. cereus) at current investigations comparable 6.25 mg/ml concentrations level [69]. These superior ability of MSL plant extracts to prevent the formation of biofilms might have been associated to intervention with organic and inorganic nutrients, as well as sedimentation, brownian, electrostatic and Van der Waals interaction factors that promote the bacterial cell growth and adhesion to surfaces [70,71]. The distinctiveness of the present research is that there have been no reports on employing MS to suppress the cell growth of bacterial biofilms yet.

Membrane stabilization potential of MSL and MSS extractives
Through the activation of lysosomal enzymes or inflammatory cytokines in stimulus to wounds, infection, or other factors (hypotonic solution) instigates one of the complex biological states termed as inflammation. This leads to engorged cell membranes, one that cause vasodilation and increased permeability of blood vessels, allowing neutrophils, fluids, and plasma protein to extravasate into the tissues along with hemolysis and hemoglobin oxidation [72,73]. Human red blood cell (HRBC) or erythrocyte membranes are nearly identical to lysosomal membranes [74] and were utilized in stabilization assays to ascertain the anti-inflammatory effects of ongoing research most prominent ethyl acetate MSL extracts ahead of addressing the issues likely arterial hypertension, stroke, heart failure, acute myocardial infarction and renal failure originated by conventional NSAIDs [75]. Ethyl acetate extracts illustrated dose dependent membrane stabilization at the concentrations of 25 -200 µg/ml (Fig. 5). Of them, at 100 µg/ml concentrations this same extract obtained 92 ± 0.2% membrane stability which explicitly paramount than standard diclofenac sodiums 57.7 ± 0.2% stability at the similar dose level. Tantary et al. (2017), Umukoro and Ashrobi (2006) carried out identical research and figured that the plant extracts they inspected attenuated RBC hemolysis by 70-90% [76,77]. By preventing the expulsion of certain protease and bactericidal enzymes like activated neutrophils lysosomal components, these plant extracts perhaps capable of stabilizing lysosomal membranes or controlling key pro-inflammatory cytokines. Numerous reports have proposed that the presence of flavonoids, terpenoids, and saponin in  plants contributes significantly to the usefulness of lysosomal membrane stability [78], as well as being consistent with our phytochemical screening investigations. This is clarified by the fact that Acheflan and Daflon are anti-inflammatory pharmaceutical flavonoid medicines with minimal side effects [72].

Molecular docking simulation of MSL antimicrobial and antiiflammatory robustness
Conducting an encyclopedic literature review to analyze 23 chemicals from MSL (Table 8), 22 of them (except Tetratetracontane) had successfully navigated possible bioavailability issues, per the rules of five taken from the 90th percentile of therapeutic candidates that advanced phase II clinical trials [79]. Because the therapeutic success of the relevant pharmacological candidates is contingent on retaining a sufficient dosage in the target location [80]. Subsequently, this therapeutic interaction for effectiveness were virtually verified employing molecular docking tools comprehensively with pathogenic organisms virulent target protein namely DNA gyrase subunit b S. aureus (PDB ID: 1KZN), LpxC of P. aeruginosa (PDB ID: 2VES), Structure of the chitin deacetylase AngCDA from Aspergillus niger (PDB ID: 7BLY) and cyclooxygenase-2 (6COX). Whereby, 1KZN-Myrtenol forming complex with three hydrogen (Val 120, Ile 90) and one hydrophobic bonds of Ala 96 residues generating lower most energy of -5.277 Kcal/mol backed by alpha-cubebene and gamma-Muurolene. This fortify affinity meanwhile conspicuously superior than contemporary drug ciprofloxacins -3.912 Kcal/mol bonding energy (Fig. 6A). Table 9 showed that UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (PDB ID: 2VES) similar sturdiest inhibition markedly prominent with T-cadinol (-4.118 Kcal/mol) constituting two H-bond (Arg 143, Glu 138) and one H-phobic (Met 162) interactions that over than reference drugs affinity (Fig. 6B). In terms of antifungal and membrane stabilization efficacy, 1, 2-Benzenedicarboxylic acid pointedly outdated the both sertaconazole and diclofenac sodium interacting energy which were furthest. Formation of three hydrogen bonds of Lys 164 and Lys 180, and six hydrophobic bonds of Lys 164, Lys 180 and Asp 162 residues (Fig. 6C) combining 1,2-Benzenedicarboxylic acid with chitin deacetylase (PDB ID: 7BLY) aiming -5.257 Kcal/mol energy whilst sertaconazole generated -4.011 Kcal/mol. Antiinflammatory mechanism therefore affirmed through quantifying modulation of cyclooxygenase-2 (PDB ID: 6COX) in which 1,2-Benzenedicarboxylic acid displayed excellent binding affinity of -6.797 Kcal/mol with originating six hydrogen and two electrostatic bonds (Fig. 6D). Details binding interaction distinctive features inscribed in Table 10. This following adjacent binding affinity in active site formulated a number variable non-covalent bonds that might be attributed to the possible inhibition of target proteins virulence factors [81]. Literature thereby dissected antimicrobial and fungicide potentialities of one key compound 1, 2-Benzenedicarboxylic acid derivatives isolated from plants [82,83].

Conclusion
This conclusive study significantly conveyed that the ethyl acetate extract of Mikania scandens leaves disclosed broad spectrum  antimicrobial properties in multitude parameters of bactericidal, fungicidal and biofilm inhibition against human pathogenic and carbapenem resistant pathogens over stem parts efficacy. Therefore membrane stabilizing modulatory effects in acute inflammation bring forth substantial vascular alterations like anti-inflammatory potency. Furthermore molecular docking assay vindicated those experimental findings precisely. These all were homologous with its traditional grandeur. Since there are a growing number bacterial strains that are resistant to conventional antibiotics, this property may be a helpful ally in the development of medications to combat this culminating microbial resistance problem. Hence, extensive mechanistic function research using these plant extract is still required to invent novel antimicrobial agents for complementary medicine.

Funding
No particular grant was given to this research by funding organizations in the public, private, or not-for-profit sectors.

Table 10
Binding interaction of MSL best compounds with antimicrobial and anti-inflammatory target proteins